1. Field of the Invention
This invention relates to derivatives, such as fragments, of toxins, particularly clostridial neurotoxins. It also relates to preparations containing those derivatives and to methods of obtaining the derivatives and the preparations.
2. Description of the Related Art
The clostridial neurotoxins are proteins with molecular masses of the order of 150 kDa. They are produced by various species of bacterium of the genus Clostridium, most importantly C. tetani and several strains of C. botulinum. There are at present eight neurotoxins known: tetanus toxin, and botulinum neurotoxin in its serotypes A, B, C1, D, E, F and G, and they all share similar structures and modes of action. The clostridial neurotoxins are synthesised by the bacterium as a single polypeptide that is modified post-translationally to form two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H), which has a molecular mass of approximately 100 kDa, and the light chain (L), which has a molecular mass of approximately 50 kDa.
The clostridial neurotoxins bind to an acceptor site (Black, J. D. & Dolly, J. O., Neuroscience, 23, 767–779, 1987 and Dolly et al. in Cellular and Molecular Basis of Cholinergic Function, ed. Dowdall, M. J. & Hawthorne, J. N., Chapter 60, 1987) on the cell membrane of the motoneurone at the neuromuscular junction and are internalised by an endocytotic mechanism (Montecucco et al., Trends Biochem. Sci., 11, 314, 1986). It is believed that the clostridial neurotoxins are highly selective for motoneurons due to the specific nature of the acceptor site on those neurones. The binding activity of clostridial neurotoxins is known to reside in a carboxy-terminal region of the heavy chain component of the dichain neurotoxin molecule, a region known as HC. The N-terminal region of the H-chain (HN domain) is thought to be of central importance in the translocation of the L-chain into the cytosol and has been demonstrated to from channels in lipid vesicles (Shone et al, Eur. J. Biochem. 167, 175–180).
Clostridial neurotoxins possess a highly specific zinc-dependent endopeptidase activity that is known to reside in the L-chain. Each toxin serotype hydrolyses a specific peptide bond within one of three proteins of the SNARE complex; VAMP (synaptobrevin), syntaxin or SNAP-25. Proteolytic cleavage of one of these proteins leads to instability of the SNARE complex and consequent prevention of vesicular release. The enzymatic activity of the light chain of the neurotoxin leads to inhibition of neurotransmitter release, which results in a prolonged muscular paralysis.
The central role of the SNARE proteins in regulated secretion has been convincingly established (e.g. Niemann et al., (1994) Trends Cell Biol., 4, 179–185). However, the correlation of SNARE protein involvement with the release of specific hormones, peptides, transmitters and other signalling molecules remains to be established in the majority of cases. The range of highly specific endopeptidase activities of clostridial neurotoxin serotypes provides a unique approach to the understanding of SNARE-mediated events. Unfortunately, the use of native clostridial toxins for the study of such events is limited by at least two important aspects. Firstly, the expression of the requisite toxin receptor is restricted to a limited population of cells, thereby limiting the range of cell types in which SNARE-mediated events can be studied without cellular disruption. Secondly, the significant hazards associated with working with potent neurotoxins lead to restrictions on the range of applications and experimental design. Clostridial neurotoxins are the most potent neuroparalytic toxins known and must be manipulated in specialised laboratory conditions by specially trained and, preferably, vaccinated staff. The ability to produce highly purified non-toxic fragments of clostridial neurotoxins possessing the enzymatic activity of the clostridial neurotoxins and capable of delivery to the cytosol of selected cells would therefore provide a valuable tool for studying secretory mechanisms.
The ability of the clostridial neurotoxins' enzymatic activity to destabilise SNARE complex formation and thereby inhibit vesicle fusion at the plasma membrane also has therapeutic potential. A number of therapeutic applications have been proposed (e.g. WO 96/33273 & WO 94/21300) that are dependent on the successful retargeting of clostridial neurotoxin fragments. These approaches require a source of non-hazardous neurotoxin fragment that is suitable for the synthesis of non-toxic conjugates, since the side effect profile of a therapeutic contaminated with neurotoxin would be unacceptably high. In addition to retargeting of clostridial toxin fragments, there are further applications for non-toxic clostridial derivatives. For example, as an immunogen for vaccine preparation, as a source of material from which highly purified neurotoxin-related fragments can be prepared, and as a non-toxic endopeptidase standard in diagnostic kits (e.g. WO 95/33850).
Since the cell binding function of clostridial neurotoxins resides in the HC domain of the heavy chain, generation of a fragment in which the binding capability of the HC has been deleted but the properties of the HN domain are retained (LHN) is potentially a suitable method for the production of a non-toxic derivative.
It is known to prepare these fragments by proteolytic treatment of toxin and then separation of toxin from fragments by anion exchange chromatography, and such methods successfully yield fragments that are 99.99% pure.
A central or recurrent problem associated with obtaining or using products from toxins made by these methods is the risk of residual toxicity in those products. It would hence be desirable to provide a method of removing toxin from such products. However, existing protocols have reached the limits of their abilities in this respect.
For example, it has been observed that the known fragments often exhibit a high inhibition of neurotransmitter release by neuronal cells in vitro. This has hampered investigation into the properties of conjugates in which a toxin fragment is combined with a ligand providing a specific targeting function, because of difficulty in providing controls against which to judge the conjugate activity.